The overall long-term objective of these studies is to develop and use transgenic animal model systems to study environmental health-related issues. Specifically, our studies focus on the metallothioneins (MTs) and transcription factors that regulate MT genes. We propose to determine the molecular mechanisms regulating expression of MT genes in response to cadmium (Cd) and oxidative stresses, and the functional significance of MT in protection from oxidative stress. Cd is a widespread environmental pollutant and many xenobiotics cause damaging oxidative stress. MTs represent the best documented intracellular heavy metal (Zn, Cu, Cd) binding proteins, but they can also scavenge hydroxyl free radicals, and dismutate superoxide anions. Transcriptional induction of the mouse MT-I gene by oxidative stress involves activation of MTF-1 binding to metal responsive elements (MRE) in the promoter, as well as a composite USF/antioxidant response promoter element (USF/ARE). Preliminary evidence indicates that Cd induction is also mediated, in part, by this element whereas Zn induction is not. Little is known about the structure and function of MTF-1 or the USF/ARE. Transgenic mice that over-express MT or that have targeted ablations of MT-I/MT-II gene function have been created and will be used to examine the roles of these proteins in protection from oxidative stress. The specific aims of this proposal are to: 1) Determine the molecular mechanisms by which oxidative stresses and metals activate MTF-1; 2) Explore the functional roles of the USF/antioxidant response element in the mouse MT-I promoter during induction by oxidative stress and Cd; 3) Examine protein interactions with the USF/ARE during oxidative stress and Cd induction of gene expression; and 4) Analyze resistance to oxidative stress in transgenic mice that over-express MT or that have targeted ablations of the MT genes. Molecular (mutagenesis, EMSA, footprinting, transfection) and biochemical (Zn titration analysis, immunoprecipitation, binding site chromatography) approaches will be used to define structure-function relationships for MTF-1 (Zn binding, DNA binding, protein interactions and transactivation). Structure-function of the USF/ARE will be defined by mutagenesis, transfection assays, and protein binding assays. Resistance to oxidative stress in transgenic mice will be monitored by histopathology and serum enzyme levels.